Method for treating a patient with neoplasia by treatment with a paclitaxel derivative

ABSTRACT

This invention provides a method for treating a patient with neoplasia by an adjuvant therapy that includes treatment with a paclitaxel derivative.

This application is a Continuation of prior U.S. application Ser. No.09/190,830 filed Oct. 12, 1998, now U.S. Pat. No. 6,235,776, entitled“Method for Treating a Patient with Neoplasia by Treatment with aPaclitaxel Derivative,” which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Virtually all of the many antineoplastic drugs that are currently usedin the treatment of cancer have very serious and harmful side effects.This is because cancer is generally treated with medications thatinterfere with the growth of rapidly dividing cells. Such medicationscan inhibit the growth of the cancer cells, but they almost always alsoinhibit the growth of normal cells that divide rapidly in the body. Someof the normal tissues that divide very rapidly include bone marrow(which produces blood cells), hair follicles, and intestinal epithelium.The usefulness of virtually all antineoplastic drugs is severely limitedby the damage they cause to these normal tissues.

This invention relates to methods for treating neoplasia using both apaclitaxel derivative (a common chemotherapeutic) and a cyclic GMP(cGMP)-specific phosphodiesterase (PDE) inhibitor to reduce the sideeffects or increase the efficacy of paclitaxel treatment. Paclitaxelderivatives (e.g., taxol) have been used to treat certain cancers,particularly ovarian and breast cancers in patients where at leastfirst-line chemotherapy has failed. Under current practice, taxoltherapy is typically used after first-line failure because taxol is sotoxic and its side effects are so bad that the risks of the therapyoutweigh the benefits until other chemotherapeutic options commonly havebeen exhausted.

The side effects of taxol include anaphylaxis and severehypersensitivity reactions characterized by dyspnea and hypotension,angiodemia, and generalized urticaria. Side effects also include nauseaand vomiting. However, the dose-limiting side effect of taxol is bonemarrow suppression. Bone marrow produces blood cells. Taxol can lowerthe number of white blood cells that guard against infections, and lowerthe number of platelets that prevent bleeding. Still other side effectsinclude neuropathy, joint and muscle pain or weakness, alopecia (orcomplete hair loss, which almost always occurs with taxol).

Investigators have reported that some of its side effects can beattenuated by the pre-medication of the cancer patient with a medicationthat eliminates or reduces hypersensitivity. For example, in U.S. Pat.No. 5,670,537, the applicants reported that if a cancer patient ispretreated with a steroid or the like, the dose of taxol between 135 and175 mg/m² can be administered in about three hours. However, thePhysicians' Desk Reference warns that even in this mode ofadministration, taxol still causes side effects severe enough to warrantits discontinuation in some patients—patients who already failed torespond to first-line therapy, of course. In other words, taxol is notrecommended for first stage cancer therapy given its side effects.

Efforts have been made to improve paclitaxel, but those efforts haveconcentrated mainly in improving its administrability (e.g., developingmore water-soluble forms of the medication). However, few, if any,investigators have reported a significantly better way to reduce theside effect of paclitaxel while maintaining its therapeutic effect, orto increase its therapeutic effect while not increasing the sideeffects.

SUMMARY OF THE INVENTION

This invention relates to an improved method of cancer therapy thatinvolves treating a patient with both a paclitaxel derivative (e.g.,taxol) and a cyclic GMP-specific phosphodiesterase (PDE) inhibitor. Thespecific PDE inhibitors useful for this invention are compounds thatinhibit both PDE5 and the new cGMP-specific PDE described below. Thenovel cGMP-PDE is fully described by Liu, et al., in the copending U.S.patent application Ser. No. 09/173,375, now U.S. Pat. No. 6,200,771, ANovel Cyclic GMP-Specific Phosphodiesterase And Methods For Using SameIn Pharmaceutical Screening For Identifying Compounds For Inhibition OfNeoplastic Lesions. (For general PDE background, see, Beavo, J. A.(1995) Cyclic nucleotide phosphodiesterases: functional implications ofmultiple isoforms. Physiological Reviews 75:725-747; web site<http://weber.u.washington.edu/˜pde/pde.html>(November 1998)).

In this invention, the cGMP-specific PDE inhibitor can be used incombination with paclitaxel in two ways. The first is a lower dosagemethodology in which the traditionally recommended dose range ofpaclitaxel is decreased while its therapeutic effects are maintained andits side effects are attenuated. The second is a higher dosagemethodology that utilizes the traditionally recommended dose range forpaclitaxel and improves its activity without increasing its sideeffects.

With either methodology, paclitaxel is administered simultaneously or insuccession with a cGMP-specific phosphodiesterase inhibitor, preferablyan inhibitor of cGMP-specific phosphodiesterases (“PDE”) found inneoplastic cells, of which there are several. It is even more preferredthat such an inhibitor have an inhibiting effect on at least severalsuch PDEs, specifically PDE5 and a new cGMP specific phosphodiesterasefound in neoplastic cells, which is described below.

In the low dose regime, paclitaxel is administered at doses less than135 mg/m² over a period of less than six hours. In the high dose regime,paclitaxel is administered at doses between about 135 and 175 mg/m² overa period of less than six hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from SW-480 neoplastic cells, as assayed from a the eluent froma DEAE-Trisacryl M column.

FIG. 2 is a graph of cGMP activities of the reloaded cGMPphosphodiesterases obtained from SW-480 neoplastic cells, as assayedfrom a the eluent from a DEAE-Trisacryl M column.

FIG. 3 is a graph of the kinetic behavior of the novel PDE.

FIG. 4 illustrates the inhibitory effects of sulindac sulfide andexisulind on PDE4 and PDE5purified from cultured tumor cells.

FIGS. 5A and 5B illustrate the effects of sulindac sulfide on cyclicnucleotide levels in HT-29 cells.

FIG. 6 illustrates the phosphodiesterase inhibitory activity of CompoundB.

FIG. 7 illustrates the phosphodiesterase inhibitory activity of CompoundE.

FIG. 8 illustrates the effects of sulindac sulfide and exisulind ontumor cell growth.

FIGS. 9A and 9B illustrate the growth inhibitory and apoptosis-inducingactivity of sulindac sulfide and control (DMSO).

FIG. 10 illustrates the growth inhibitory activity of compound E.

FIGS. 11A and 11B illustrate the effects of sulindac sulfide andexisulind on apoptosis and necrosis of HT-29 cells.

FIGS. 12A and 12B illustrate the effects of sulindac sulfide andexisulind on HT-29 cell growth inhibition and apoptosis induction asdetermined by DNA fragmentation.

FIG. 13 illustrates the apoptosis inducing properties of Compound E.

FIG. 14 illustrates the apoptosis inducing properties of Compound B.

FIG. 15 illustrates the inhibition of pre-malignant, neoplastic lesionsin mouse mammary gland organ culture by sulindac metabolites.

FIG. 16 illustrates the effects of paclitaxel and exisulind (FGN-1) ontumor cell growth at 25 μM exisulind.

FIG. 17 illustrates the effects of paclitaxel and exisulind (FGN-1) ontumor cell growth at 50 μM exisulind.

FIG. 18 illustrates the effects of paclitaxel and exisulind (FGN-1) ontumor cell growth at 100 μM exisulind.

FIG. 19 illustrates the effects of paclitaxel and exisulind (FGN-1) ontumor cell growth at 200 μM exisulind.

FIG. 20 illustrates the effects of paclitaxel and exisulind (FGN-1) ontumor cell growth at 400 μM exisulind.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As discussed in greater detail below, the inhibition of cGMP-specificPDEs can induce apoptosis in neoplastic cells. Paclitaxel derivativesare currently used to treat neoplasias, particularly breast and ovariancancers. The combination of these two types of therapies can produce aneffect that neither can produce n individually.

I. The Novel cGMP-Specific Phosphodiesterase

A new cyclic GMP-specific phosphodiesterase has been discovered inneoplastic cells. Treatment of cells with a compound that inhibits bothPDE5 and this novel cGMP-specific PDE leads to apoptosis of theneoplastic cells. In other words, the preferred cGMP-specific inhibitorsuseful in this invention, in combination with paclitaxel are thosecompounds that inhibit both PDE5 and this new PDE.

The new PDE is broadly characterized by

(a) cGMP specificity over cAMP;

(b) positive cooperative kinetic behavior in the presence of cGMPsubstrate;

(c) submicromolar affinity for cGMP; and

(d) insensitivity to incubation with purified cGMP-dependent proteinkinase.

As discussed below, this new cGMP-PDE is unique from the classical PDE5.Kinetic data reveal that the new PDE has increased cGMP hydrolyticactivity in the presence of increasing cGMP substrate concentrations,unlike PDE5 which exhibits cGMP substrate saturation. The new cGMP-PDEis insensitive to incubation with cGMP-dependent protein kinase (PKG),whereas PDE5 is phosphorylated by PKG. Additionally, the new cGMP-PDE isrelatively insensitive to inhibition with the PDE5-specific inhibitors,zaprinast and E4021. Finally, the new cGMP-PDE activity can be separatedfrom classical PDE5 activity by anion-exchange chromatography.

The new cGMP-PDE is not a member of any of the other previouslycharacterized PDE families. The new PDE does not hydrolyze cAMPsignificantly. Calcium (with or without calmodulin) failed to activateeither Ms cAMP or cGMP hydrolysis activity, indicating that the novelPDE is not a CaM-PDE (PDE1). Additionally, cGMP failed to activate orinhibit cAMP hydrolysis, indicating that the new cGMP-PDE it is not acGMP-stimulated PDE (cGS-PDE or PDE2), because all known isoforms of thePDE2 family hydrolyze both cAMP and cGMP. Further, the new cGMP-PDE isinsensitive to a number of specific PDE inhibitors. It is relativelyinsensitive to vinpocetine (a CaM-PDE- or PDE1-specific inhibitor), toindolodan (a cGI-PDE- or PDE3-specific inhibitor), and to rolipram (acAMP-PDE- or PDE4-specific inhibitor). The data establish that the newPDE is not a member of one of the cAMP-hydrolyzing PDE families (PDE1,PDE2, PDE3, or PDE4).

PDE inhibitors that are useful for treating patients with neoplasiaconsistent with this invention should inhibit both PDE5 and the newcGMP-PDE. A compound that inhibits both forms of cGMP-specific PDE isdesirable because a compound that inhibits PDE5 but not the new PDE,does not by itself induce apoptosis. For example, zaprinast, sildenafil,and E4021 have been reported as potent inhibitors of PDE5. However,compared to PDE5, the new PDE is relatively insensitive to zaprinast,sildenafil, and E4021 (Table 1). And none of the three, zaprinast,sildenafil, or E4021, have been found to induce apoptosis (Table 6) orto inhibit cell growth in neoplastic cells (Tables 3 and 4).

However, a number of PDE5 inhibitors have been found to induce apoptosisin neoplastic cells. Examples of such compounds are sulindac sulfide andCompound E. Sulindac sulfide and Compound E each inhibit PDE5 and thenew cGMP-PDE with the same potency (Table 1). And both sulindac sulfideand Compound E induce apoptosis in neoplastic cells (Table 6). Compoundsthat inhibit PDE5, but not the new cGMP-PDE, do not cause apoptosis inneoplastic cells. But compounds that inhibit both PDE5 and the newcGMP-PDE, have been found to induce apoptosis in neoplastic cells.

A. Isolation of the Novel cGMP-Specific Phosphodiesterase

The novel cGMP-specific phosphodiesterase can be isolated from humancarcinoma cell lines (e.g. SW-480, a human colon cancer cell line thatoriginated from a moderately differentiated epithelial adenocarcinoma,available from the American Tissue Type Collection in Rockville, Md.,U.S.A.). The complete isolation of this new cGMP-PDE is described in thecopending application, Liu, et al., U.S. patent application Ser. No.09/173,375, now U.S. Pat. No. 6,200,771, A Novel Cyclic GMP-SpecificPhosphodiesterase And Methods For Using Same In Pharmaceutical ScreeningFor Identifying Compounds For Inhibition Of Neoplastic Lesions, which isincorporated herein by reference.

Briefly, to isolate the novel phosphodiesterase, SW-480 cells arecollected and homogenized. The homogenate is centrifuged, and thesupernatant is loaded onto a DEAE-Trisacryl M column. The loaded columnis then washed, and PDE activities are eluted with a linear gradient ofNaOAc. Fractions are collected and immediately assayed for cGMPhydrolysis activity. Cyclic nucleotide PDE activity of each fraction isdetermined using the modified two-step radioisotopic method of Thompsonet al. (Thompson W. J., et al., Adv Cyclic Nucleotide Res 10: 69-92,1979). There are two initial peaks of cGMP-PDE activity eluted from thecolumn, peak A and peak B (see FIG. 1). Peak A is PDE5, whereas peak Bis the new cGMP-PDE.

To further fractionate the cGMP hydrolytic activity of PDE5 and the newcGMP-PDE, the fractions containing those activities are reloaded ontothe DEAE-Trisacryl M column and eluted with a linear gradient of NaOAc.Fractions are again immediately assayed for cGMP hydrolysis activity,the results of which are presented in FIG. 2. As illustrated in FIG. 2,peak B, the novel PDE, shows enhanced activity with increasing cGMPsubstrate concentration. Peak A, on the other hand, shows apparentsubstrate saturation with increasing concentrations of cGMP.

B. cGMP-Specificity of PDE Peaks A and B

Each fraction from the DEAE column was also assayed for cGMP-hydrolysisactivity (0.25 μM cGMP) in the presence or absence of Ca⁺⁺, or Ca⁺⁺-CaMand/or EGTA and for cAMP (0.25 μM cAMP) hydrolysis activity in thepresence or absence of 5 μM cGMP. Neither PDE peak A nor peak B(fractions 5-22; see FIG. 1) hydrolyzed cAMP significantly, establishingthat neither was a member of a cAMP hydrolyzing family of PDEs (i.e. aPDE 1, 2, 3).

Ca⁺⁺ (with or without calmodulin) failed to activate either cAMP or cGMPhydrolysis activity of either peak A or B, and cGMP failed to activateor inhibit cAMP hydrolysis. Such results establish that peaks A and Bconstitute cGMP-specific PDEs but not PDE1, PDE2, PDE3, or PDE4.

For PDE peak B, as discussed below, cyclic GMP activated the cGMPhydrolytic activity of the enzyme, but did not activate any cAMPhydrolytic activity. This reveals that PDE peak B—the novelphosphodiesterase—is not a cGMP-stimulated cyclic nucleotide PDE (“cGS”)or among the PDE2 family isoforms because the known isoforms of PDE2hydrolyze both cGMP and cAMP.

C. Peak A is a PDE5, But Peak B—a New cGMP-Specific PDE—Is Not

To characterize any PDE isoform, kinetic behavior and substratepreference should be assessed. Peak A showed typical “PDE5”characteristics. For example, the K_(m) of the enzyme for cGMP was 1.07μM, and Vmax was 0.16 nmol/min/mg. In addition, as discussed below,zaprinast (IC₅₀=1.37 μM), E4021 (IC₅₀=3 nM), and sildenafil inhibitedactivity of peak A. Further, zaprinast showed inhibition for cGMPhydrolysis activity of peak A, consistent with results reported in theto literature for PDE5.

PDE peak B showed considerably different kinetic properties as comparedto PDE peak A. For example, in Eadie-Hofstee plots of peak A, cyclic GMPhydrolysis shows a single line with negative slope with increasingsubstrate concentrations, indicative of Michaelis-Menten kineticbehavior. Peak B, however, shows the novel property for cGMP hydrolysisin the absence of cAMP of a decreasing (apparent K_(m)=8.4), thenincreasing slope (K_(m)<1) of Eadie-Hotfstee plots with increasing cGMPsubstrate (see FIG. 3). This establishes peak B's submicromolar affinityfor cGMP (i.e., where K_(m)<1).

Consistent with the kinetic studies (i.e., FIG. 3) andpositive-cooperative kinetic behavior in the presence of cGMP substrate,was the increased cGMP hydrolytic activity in the presence of increasingconcentrations of cGMP substrate. This was discovered by comparing 0.25μM, 2 μM, and 5 μM concentrations of cGMP in the presence of PDE peak Bafter a second DEAE separation to rule out cAMP hydrolysis and to ruleout this new enzyme being a “classic” PDE5. Higher cGMP concentrationsevoked disproportionately greater cGMP hydrolysis with PDE peak B, asshown in FIG. 2.

These observations suggest that cGMP binding to the peak B enzyme causesa conformational change in the enzyme.

D. Zaprinast- and Sildenafil-Insensitivity of PDE Peak B Relative toPeak A, and Their Effects on Other PDE Inhibitors

Different PDE inhibitors were studied using twelve concentrations ofdrug from 0.01 to 100 μM and substrate concentration of 0.25 μM ³H-cGMP.IC₅₀ values were calculated with variable slope, sigmoidal curve fitsusing Prism 2.01 (GraphPad). The results are shown in Table 1. Whilecompounds E4021 and zaprinast inhibited peak A, (with high affinities)IC₅₀ values calculated against peak B are significantly increased (>50fold). This confirms that peak A is a PDE5. These data furtherillustrate that the novel PDE is, for all practical purposes,zaprinast-insensitive and E4021-insensitive.

TABLE 1 Comparison of PDE Inhibitors Against Peak A and Peak B (cGMPHydrolysis) PDE Family IC₅₀ IC₅₀ Ratio (IC₅₀ Compound Inhibitor Peak A(μM) Peak B (μM) PeakA/Peak B) E4021 5 0.003 8.4 0.0004 Zaprinast 51.4 >30 <0.05 Compound E 5 and others 0.38 0.37 1.0 Sulindac 5 andothers 50 50 1.0 sulfide Vinpocetine 1 >100 >100 EHNA 2,5 >100 3.7Indolidan 3 31 >100 <0.31 Rolipram 4 >100 >100 Sildenafil 5 .0003 >10<.00003

By contrast, sulindac sulfide and Compound E competitively inhibit bothpeak A and peak B phosphodiesterases at the same potency (for CompoundE, IC₅₀=0.38 μM for PDE peak A; IC₅₀=0.37 μM for PDE peak B).

There is significance for the treatment of neoplasia and the selectionof useful compounds for such treatment in the fact that peak B iszaprinast-insensitive whereas peaks A and B are both sensitive tosulindac sulfide and Compound E. Zaprinast, E4021, and sildenafil havebeen tested to ascertain whether they induce apoptosis or inhibit thegrowth of neoplastic cells, and the same has been done for Compound E.As explained below, zaprinast, sildenafil and E4021 do not havesignificant apoptosis-inducing (Table 6) or growth-inhibiting (Tables 3and 4) properties, whereas sulindac sulfide and Compound E are preciselythe opposite. In other words, the ability of a compound to inhibit bothPDE peaks A and B correlates with its ability to induce apoptosis inneoplastic cells, whereas if a compound (e.g., zaprinast) hasspecificity for PDE peak A only, that compound will not induceapoptosis.

E. Insensitivity of PDE Peak B to Incubation With cGMP-Dependent ProteinKinase

Further differences between PDE peaks A and B were observed in theirrespective cGMP-hydrolytic activities in the presence of varyingconcentrations of cGMP-dependent protein kinase (PKG, whichphosphorylates typical PDE5). Specifically, peak A and peak B fractionswere incubated with different concentrations of protein kinase G at 30°C. for 30 minutes. Cyclic GMP hydrolysis of both peaks was assayed afterphosphorylation was attempted. Consistent with previously publishedinformation about PDE5, peak A showed increasing cGMP hydrolysisactivity in response to protein kinase G incubation, indicating thatpeak A was phosphorylated. Peak B was unchanged, however (i.e., was notphosphorylated and was insensitive to incubation with cGMP-dependentprotein kinase). These data are consistent with peak A being a PDE5family isoform and peak B being a novel cGMP-DE.

II. Selecting a cGMP-Specific Phosphodiesterase Inhibitor for Use inthis Invention

Cancer and precancer may be thought of as diseases that involveunregulated cell growth. Cell growth involves a number of differentfactors. One factor is how rapidly cells proliferate, and anotherinvolves how rapidly cells die. Cells can die either by necrosis orapoptosis depending on the type of environmental stimuli. Celldifferentiation is yet another factor that influences tumor growthkinetics. Resolving which of the many aspects of cell growth is affectedby a test compound is important to the discovery of a relevant targetfor pharmaceutical therapy. Assays based on this technology can becombined with other tests to determine which compounds have growthinhibiting and proapoptotic activity.

In this invention, particular cGMP-specific PDE inhibitors are selectedfor use in combination with paclitaxel to treat neoplasia, particularlybreast cancer and ovarian cancer which are commonly treated withpaclitaxel. The combination therapy can be implemented in one of severalways.

As indicated above, preferred PDE inhibitors are those that inhibit theactivities of both PDE5 and the new cGMP-PDE. A compound can be selectedfor use in this invention by evaluating its effect on the cGMPhydrolytic activity on a mixture of the two enzymes (i.e., a mixture ofpeaks A and B) isolated from a tumor cell line. Alternatively, acompound can be selected by evaluating the compound's effect on cyclicnucleotide levels in whole neoplastic cells before and after exposure ofthe cells to the compound of interest. Still another alternative is totest a compound of interest against the two PDEs separately, i.e., byphysically separating each activity from a tumor cell line (or by usingrecombinant versions of each enzyme) and testing the inhibitory actionof the compound against each enzyme individually. With any of the aboveapproaches, an appropriate PDE inhibitor can be selected for use incombination with paclitaxel.

A. Phosphodiesterase Enzyme Assay

Phosphodiesterase activity (whether in a mixture or separately) can bedetermined using methods known in the art, such as a method using aradioactively labeled form of cGMP as a substrate for the hydrolysisreaction. Cyclic GMP labeled with tritium (³H-cGMP) is used as thesubstrate for the PDE enzymes. (Thompson, W. J., Teraski, W. L.,Epstein, P. M., Strada, S. J., Advances in Cyclic Nucleotide Research,10:69-92, 1979, which is incorporated herein by reference). In thisassay, cGMP-PDE activity is determined by quantifying the amount of cGMPsubstrate that is hydrolyzed either in the presence or absence of thetest compound).

In brief, a solution of defined substrate ³H-cGMP specific activity ismixed with the drug to be tested. The mixture is incubated with isolatedPDE activity (either a single PDE or a mixture of PDE activities). Thedegree of phosphodiesterase inhibition is determined by calculating theamount of radioactivity released in drug-treated reactions and comparingthose against a control sample (a reaction mixture lacking the testedcompound but with the drug solvent).

B. Cyclic Nucleotide Measurements

Alternatively, the ability of a compound to inhibit cGMP-PDE activity isreflected by an increase in the levels of cGMP in neoplastic cellsexposed to the test compound. The amount of PDE activity can bedetermined by assaying for the amount of cyclic GMP in the extract oftreated cells using a radioimrnmunoassay (RIA). In this procedure, aneoplastic cell line is incubated with a test compound. After about 24to 48 hours, the cells are solubilized, and cyclic GMP is purified fromthe cell extracts. The cGMP is acetylated according to publishedprocedures, such as using acetic anhydride in triethylamine, (Steiner,A. L., Parker, C. W., Kipnis, D. M., J. Biol. Chem., 247(4):1106-13,1971, which is incorporated herein by reference). The acetylated cGMP isquantitated using radioimmunoassay procedures (Harper, J., Brooker, G.,Advances in Nucleotide Research, 10:1-33, 1979, which is incorporatedherein by reference).

In addition to observing increases in the content of cGMP in neoplasticcells as a result of incubation with certain test compounds, decreasesin the content of cAMP have also been observed. It has been observedthat a compound which is useful in the practice of this invention (i.e.,one that selectively induces apoptosis in neoplastic cells, but notsubstantially in normal cells) follows a time course consistent withcGMP-specific PDE inhibition. Initially, the result is an increased cGMPcontent within minutes, and secondarily, there is a decreased cAMPcontent within 24 hours. The intracellular targets of these drug actionsare being studied further, but current data support the concept that theinitial rise in cGMP content and the subsequent fall in cAMP contentprecede apoptosis in neoplastic cells exposed to test compounds usefulin this invention. To determine the cyclic AMP content in cell extracts,radioimmunoassay techniques similar to those described above for cGMPare used.

The change in the ratio of the two cyclic nucleotides may be a moreaccurate tool for evaluating cGMP-specific phosphodiesterase inhibitionactivity of test compounds, rather than measuring only the absolutevalue of cGMP, only the level of cGMP hydrolysis, or only cGMP-specificphosphodiesterase inhibition. In neoplastic cells not treated withanti-neoplastic compounds, the ratio of cGMP content/cAMP content is inthe 0.03-0.05 range (i.e., 300-500 fmol/mg protein cGMP content over6000-8000 fmol/mg protein cAMP content). After exposure to desirableanti-neoplastic compounds, that ratio increases several fold (preferablyat least about a three-fold increase) as the result of an initialincrease in cyclic GMP and the later decrease in cyclic AMP.

Specifically, it has been observed that particularly desirable compoundsachieve an initial increase in cGMP content in treated neoplastic cellsto a level of cGMP greater than about 500 fmol/mg protein. In addition,particularly desirable compounds cause the later decrease in cAMPcontent in treated neoplastic cells to a level of cAMP less than about4000 fmol/mg protein.

Verification of the cyclic nucleotide content may be obtained bydetermining the turnover or accumulation of cyclic nucleotides in intactcells. To measure the levels of cAMP in intact cells, ³H-adenineprelabeling is used according to published procedures (Whalin M. E., R.L. Garrett Jr., W. J. Thompson, and S. J. Strada, “Correlation ofcell-free brain cyclic nucleotide phosphodiesterase activities to cyclicAMP decay in intact brain slices”, Sec. Mess. and Phos. ProteinResearch, 12:311-325, 1989, which is incorporated herein by reference).The procedure measures flux of labeled ATP to cyclic AMP and can be usedto estimate intact cell adenylate cyclase or cyclic nucleotidephosphodiesterase activities depending upon the specific protocol.Cyclic GMP accumulation was too low to be studied with intact cellprelabeling according to published procedures (Reynolds, P. E., S. J.Strada and W. J. Thompson, “Cyclic GMP accumulation in pulmonarymicrovascular endothelial cells measured by intact cell prelabeling,”Life Sci., 60:909-918, 1997, which is incorporated herein by reference).

C. Tissue Sample Assay

The cGMP-specific PDE inhibitory activity of a test compound can also bedetermined from a tissue sample. Tissue biopsies from humans or tissuesfrom anesthetized animals are collected from subjects exposed to thetest compound. Briefly, a sample of tissue is homogenized and a knownamount of the homogenate is removed for protein analysis. From theremaining homogenate, the protein is allowed to precipitate. Next, thehomogenate is centrifuged and both the supernatant and the pellet arerecovered. The supernatant is assayed for the amount of cyclicnucleotides present using RIA procedures as described above.

D. Experimental Results

1. Introduction

The amount of cGMP-specific inhibition is determined by comparing theactivity of the cGMP-specific PDEs in the presence and absence of thetest compound. Inhibition of cGMP-PDE activity is indicative that thecompound is useful for treating neoplasia in combination withpaclitaxel. Significant inhibitory activity, greater than that of thebenchmark, exisulind, and preferably greater than 50% at a concentrationof 10 μM or below, is indicative that a compound should be furtherevaluated for antineoplastic properties. The term “exisulind” means(Z)-5-fluoro-2-methyl-1-[[4-(methylsulfonyl)phenyl]methylene]indene-3-ylacetic acid or a salt thereof. (See, Pamukcu and Brendel, U.S. Pat. No.5,401,774.)

2. cGMP-PDE Inhibition Assay

Reference compounds and test compounds were analyzed for their cGMP-PDEinhibitory activity in accordance with the protocol for the assaydescribed supra. FIG. 4 shows the effect of various concentrations ofsulindac sulfide and exisulind on either PDE4 or cGMP-PDE activitypurified from human colon HT-29 cultured tumor cells, as describedpreviously (W. J. Thompson et al., supra). The IC₅₀ value of sulindacsulfide for inhibition of PDE4 was 41 μM, and for inhibition of cGMP-PDEwas 17 μm. The IC₅₀ value of exisulind for inhibition of PDE4 was 181μM, and for inhibition of cGMP-PDE was 56 μM. These data show that bothsulindac sulfide and exisulind inhibit phosphodiesterase activity. Bothcompounds show selectivity for the cGMP-PDE isoenzyme forms over PDE4isoforms.

FIG. 5 shows the effects of sulindac sulfide on either cGMP or cAMPproduction as determined in cultured HT-29 cells in accordance with theassay described, supra. HT-29 cells were treated with sulindac sulfidefor 30 minutes and cGMP or cAMP was measured by conventionalradioimrnunoassay method. As indicated, sulindac sulfide increased thelevels of cGMP by greater than 50% with an EC₅₀ value of 7.3 μM (FIG.5A, top). Levels of cAMP were unaffected by treatment, although a knownPDE4 inhibitor, rolipram, increased cAMP levels (FIG. 5B, bottom). Thedata demonstrate the pharmacological significance of inhibitingcGMP-PDE, relative to PDE4.

FIG. 6 shows the effect of the indicated dose of test Compound B,described below, on either cGMP-PDE or PDE4 isozymes ofphosphodiesterase. The calculated IC₅₀ value was 18 μM for cGMP-PDE and58 μM for PDE4.

FIG. 7 shows the effect of the indicated dose of test Compound E,described below, on either PDE4 or cGMP-PDE. The calculated IC₅₀ valuewas 0.08 μM for cGMP-PDE and greater than 5 μM for PDE4.

COMPOUNDS

A number of compounds were examined in the various protocols andscreened for potential use in treating neoplasia. The results of thesetests are reported below. The test compounds are hereinafter designatedby a letter code that corresponds to the following:

A-rac-threo-(E)-1-(N,N′-diethylaminoethanethio)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;

B-(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-acetic acid;

C-(Z)-5-Fluoro-2-methyl-1-(p-chlorobenzylidene)-3-acetic acid;

D-rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-N-acetylcysteine;

E-(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetarnide,N-benzyl;

F-(Z)-5-Fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetamide,N,N′-dicyclohexyl;

G-ribo-(E)-1-Triazolo-[2′,3′:1″,3″]-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;and

H-rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-glutathione).

TABLE 2 cGMP PDE Inhibitory Activity Among a Series of CompoundsReference compounds % Inhibition at 10 μM Indomethacin 34 MY5445 86Sulindac sulfide 97 Exisulind 39 Test compounds % Inhibition at 100 μM A<25 B <25 C <25 D 36 E 75

The above compounds in Table 2 were evaluated for PDE inhibitoryactivity in HT-29 cells, as described in the protocol, supra. Of thecompounds that did not inhibit COX, only Compound E was found to causegreater than 50% inhibition at 10 μM. As noted in FIG. 6, Compound Bshowed inhibition of greater than 50% at a dose of 20 μM. Therefore,depending on the dosage level used in a single dose test, some compoundsmay be screened out that otherwise may be active at slightly higherdosages. The dosage used is subjective and may be lowered to identifyeven more potent compounds after active compounds are found at certainconcentration levels.

III. Determining Whether a Compound Reduces the Number of Tumor Cells

In an alternate embodiment, the preferred cGMP-specific inhibitorsuseful in the practice of this invention are selected by furtherdetermining whether the compound reduces the growth of tumor cells invitro. Various cell lines can be used depending on the tissue to betested. For example, these cell lines include: SW-480—colonicadenocarcinoma; HT-29—colonic adenocarcinoma; A-427—lung adenocarcinoma;MCF-7—breast adenocarcinoma; UACC-375-melanoma line; and DU145—prostratecarcinoma. Cytotoxicity data obtained using these cell lines areindicative of an inhibitory effect on neoplastic lesions. These celllines are well characterized, and are used by the United States NationalCancer Institute in their screening program for new anti-cancer drugs.

A. Tumor Inhibition in the HT-29 Cell Line

A compound's ability to inhibit tumor cell growth can be measured usingthe HT-29 human colon carcinoma cell line obtained from ATCC (Bethesda,Md.). HT-29 cells have previously been characterized as a relevant colontumor cell culture model (Fogh, J., and Trempe, G. In: Human Tumor Cellsin Vitro, J. Fogh (ed.), Plenum Press, New York, pp. 115-159, 1975).Briefly, after being grown in culture, HT-29 cells are fixed by theaddition of cold trichloroacetic acid. Protein levels are measured usingthe sulforhodamine B (SRB) colorimetric protein stain assay aspreviously described by Skehan, P., Storeng, R., Scudiero, D., Monks,A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S.,and Boyd, M. R., “New Colorimetric Assay For Anticancer-Drug Screening,”J. Natl. Cancer Inst., 82: 1107-1112, 1990, which is incorporated hereinby reference.

In addition to the SRB assay, a number of other methods are available tomeasure growth inhibition and could be substituted for the SRB assay.These methods include counting viable cells following trypan bluestaining, labeling cells capable of DNA synthesis with BrdU orradiolabeled thymidine, neutral red staining of viable cells, or MTTstaining of viable cells.

B. Experimental Results

1. Introduction

Significant tumor cell growth inhibition, greater than about 50% at adose of 100 μM or below is further indicative that the compound isuseful for treating neoplastic lesions. Preferably, an IC₅₀ value isdetermined and used for comparative purposes. This value is theconcentration of drug needed to inhibit tumor cell growth by 50%relative to the control. Preferably, the IC₅₀ value should be less than100 μM for the compound to be considered useful for treating neoplasticlesions in combination with paclitaxel according to the method of thisinvention.

2. Growth inhibition assay

Reference compounds and test compounds were analyzed for their cGMP-PDEinhibitory activity in accordance with the protocol for the assay,supra. FIG. 8 shows the inhibitory effect of various concentrations ofsulindac sulfide and exisulind on the growth of HT-29 cells. HT-29 cellswere treated for six days with various doses of exisulind (triangles) orsulindac sulfide (squares) as indicated. Cell number was measured by asulforhodamine assay as previously described (Piazza et al., CancerResearch, 55: 3110-3116, 1995). The IC₅₀ value for sulindac sulfide wasapproximately 45 μM and for exisulind was approximately 200 μM. The datashow that both sulindac sulfide and exisulind are capable of inhibitingtumor cell growth.

FIG. 9 shows the growth inhibitory and apoptosis-inducing activity ofsulindac sulfide. A time course experiment is shown involving HT-29cells treated with either vehicle, 0.1% DMSO (open symbols) or sulindacsulfide, 120 μM (closed symbols). Growth inhibition (FIG. 9A, top) wasmeasured by counting viable cells after trypan blue staining. Apoptosis(FIG. 9B, bottom) was measured by morphological determination followingstaining with acridine orange and ethidium bromide as describedpreviously (Duke and Cohen, In: Current Protocols in Immunology,3.17.1-3.17.16, New York, John Wiley and Sons, 1992). The datademonstrate that sulindac sulfide is capable of inhibiting tumor cellgrowth and that the effect is accompanied by an increase in apoptosis.All data were collected from the same experiment.

FIG. 10 shows the growth inhibitory activity of test Compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof Compound E for six days and cell number was determined by the SRBassay. The calculated IC₅₀ value was 0.04 μM.

TABLE 3 Growth Inhibitory Activity Among a Series of Compounds %Inhibition at 100 μM Reference Compound Indomethacin 75 MY5445 88Sulindac sulfide 88 Exisulind <50 E4021 <50 sildenafil <50 zaprinast <50Test compounds A 68 B 77 C 80 D 78 E 62

In accordance with the screening protocol, supra, Compounds A through Ewere tested for growth inhibitory activity, as reported in Table 3above. All the test compounds showed activity exceeding the benchmarkexisulind at a 100 μM single dose test.

The growth inhibitory activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 4 below. HT-29cell were treated for 6 days with various inhibitors ofphosphodiesterase. Cell growth was determined by the SRB assaydescribed, supra. The data below taken with those above show thatinhibitors of the cGMP-specific PDE activity were effective forinhibiting tumor cell growth.

TABLE 4 Growth Inhibitory Data for PDE Inhibitors Growth inhibitionInhibitor Reported Selectivity (IC₅₀, μM) 8-methoxy-IBMX PDE1 >200 μMMilrinone PDE3 >200 μM RO-20-1724 PDE4 >200 μM MY5445 PDE5   5 μM IBMXNon-selective >100 μM Zaprinast PDE5 >100 μM Sildenafil PDE5 >100 μME4021 PDE5 >100 μM

To show the effectiveness of cGMP-specific PDE inhibition on variousforms of neoplasia, compounds were tested on numerous cell lines. Theeffects of sulindac sulfide and exisulind on various cell lines wasdetermined. The data are shown in Table 5 below. The IC₅₀ values weredetermined by the SRB assay. The data show the effectiveness of thesecompounds on a broad range of neoplasias, with effectiveness atcomparable dose range. Therefore, compounds selected for cGMP-specificPDE inhibition in combination with paclitaxel should be useful fortreating neoplasia, in particular breast and ovarian cancers.

TABLE 5 Growth Inhibitory Data of Various Cell Lines IC₅₀ (μM)* CellType/ Sulindac Com- Tissue specificity sulfide Exisulind pound E HT-29,Colon 60 120 0.10 HCT116, Colon 45 90 MCF7/S, Breast 30 90 UACC375,Melanoma 50 100 A-427, Lung 90 130 Bronchial Epithehal Cells 30 90 NRK,Kidney (non ras-transformed) 50 180 KNRK, Kidney (ras transformed) 60240 Human Prostate Carcinoma PC3 82 0.90 Colo 205 1.62 DU-145 0.10HCT-15 0.60 MDA-MB-231 0.08 MDA-MB-435 0.04 *Determined by neutral redassay as described by Schmid et al., in Proc. AACR Vol 39, p. 195(1998).

IV. Determining Whether a Compound Induces Apoptosis

In a second alternate embodiment, preferably, the cGMP-specific PDEinhibitors useful in combination with paclitaxel in the practice of thisinvention induce apoptosis in cultures of tumor cells.

Two distinct forms of cell death may be described by morphological andbiochemical criteria: necrosis and apoptosis. Necrosis is accompanied byincreased permeability of the plasma membrane; the cells swell and theplasma membrane ruptures within minutes. Apoptosis is characterized bymembrane blebbing, condensation of cytoplasm, and the activation ofendogenous endonucleases.

Apoptosis occurs naturally during normal tissue turnover and duringembryonic development of organs and limbs. Apoptosis also is induced bycytotoxic T-lymphocytes and natural killer cells, by ionizing radiation,and by certain chemotherapeutic drugs. Inappropriate regulation ofapoptosis is thought to play an important role in many pathologicalconditions including cancer, AIDS, Alzheimer's disease, etc. CyclicGMP-specific PDE inhibitors useful in this invention can be selectedbased on their ability to induce apoptosis in cultured tumor cellsmaintained under conditions as described above.

Treatment of cells with test compounds involves either pre- orpost-confluent cultures and treatment for two to seven days at variousconcentrations of the compound in question. Apoptotic cells are measuredby combining both the attached and “floating” compartments of thecultures. The protocol for treating tumor cell cultures with sulindacand related compounds to obtain a significant amount of apoptosis hasbeen described in the literature. (See, Piazza, G. A., et al., CancerResearch, 55:3110-16, 1995, which is incorporated herein by reference).The novel features include collecting both floating and attached cells,identification of the optimal treatment times and dose range forobserving apoptosis, and identification of optimal cell cultureconditions.

A. Analysis of Apoptosis by Morphological Observation

Following treatment with a test compound, cultures can be assayed forapoptosis and necrosis by fluorescent microscopy following labeling withacridine orange and ethidium bromide. The method for measuring apoptoticcell number has previously been described by Duke & Cohen,“Morphological And Biochemical Assays Of Apoptosis,” Current ProtocolsIn Immunology, Coligan et al., eds., 3.17.1-3.17.16 (1992, which isincorporated herein by reference).

For example, floating and attached cells can be collected, and aliquotsof cells can be centrifuged. The cell pellet can then be resuspended inmedia and a dye mixture containing acridine orange and ethidium bromide.The mixture can then be examined microscopically for morphologicalfeatures of apoptosis.

B. Analysis of Apoptosis by DNA Fragmentation

Apoptosis can also be quantified by measuring an increase in DNAfragmentation in cells which have been treated with test compounds.Commercial photometric EIA for the quantitative in vitro determinationof cytoplasmic histone-associated-DNA-fragments (mono- andoligonucleosomes) are available (Cell Death Detection ELISA^(okys), Cat.No. 1,774,425, Boehringer Mannheim). The Boehringer Mannheim assay isbased on a sandwich-enzyme-immunoassay principle using mouse monoclonalantibodies directed against DNA and histones, respectively. This allowsthe specific determination of mono- and oligonucleosomes in thecytoplasmic fraction of cell lysates.

According to the vendor, apoptosis is measured in the following fashion.The sample (cell-lysate) is placed into a streptavidin-coated microtiterplate (“MTP”). Subsequently, a mixture of anti-histone-biotin andanti-DNA peroxidase conjugate are added and incubated for two hours.During the incubation period, the anti-histone antibody binds to thehistone-component of the nucleosomes and simultaneously fixes theimmunocomplex to the streptavidin-coated MTP via its biotinylation.Additionally, the anti-DNA peroxidase antibody reacts with the DNAcomponent of the nucleosomes. After removal of unbound antibodies bywashing, the amount of nucleosomes is quantified by the peroxidaseretained in the immunocomplex. Peroxidase is determined photometricallywith ABTS7 (2,2′-Azido-[3-ethylbenzthiazolin-sulfonate]) as substrate.

Fold stimulation (FS=OD_(max)/OD_(veh)), an indicator of apoptoticresponse, is determined for each compound tested at a givenconcentration. EC₅₀ values may also be determined by evaluating a seriesof concentrations of the test compound.

C. Experimental Results

1. Introduction

Statistically significant increases of apoptosis (i.e., greater than 2fold stimulation at a concentration of 100 μM) are further indicativethat the cGMP-specific PDE inhibitor is useful in combination withpaclitaxel in the practice of this invention. Preferably, the EC₅₀ valuefor apoptotic activity should be less than 100 μM for the compound to befurther considered for potential use for treating neoplastic lesions.EC₅₀ is herein defined as the concentration that causes 50% induction ofapoptosis relative to vehicle treatment.

2. Apoptosis Assay

Reference compounds and test compounds were analyzed for theircGMP-specific PDE inhibitory activity in accordance with the protocolsfor the assay, supra. In accordance with those protocols, FIG. 11 showsthe effects of sulindac sulfide and exisulind on apoptotic and necroticcell death. HT-29 cells were treated for six days with the indicateddose of either sulindac sulfide or exisulind. Apoptotic and necroticcell death was determined as previously described (Duke and Cohen, In:Current Protocols in Immunology, 3.17.1-3.17.16, New York, John Wileyand Sons, 1992). The data show that both sulindac sulfide and exisulindare capable of causing apoptotic cell death without inducing necrosis.All data were collected from the same experiment.

FIG. 12 shows the effect of sulindac sulfide and exisulind on tumorgrowth inhibition and apoptosis induction as determined by DNAfragmentation. The top figure (12A) shows growth inhibition (opensymbols, left axis) and DNA fragmentation (closed symbols, right axis)by exisulind. The bottom figure (12B) shows growth inhibition (opensymbols) and DNA fragmentation (closed symbols) by sulindac sulfide.Growth inhibition was determined by the SRB assay after six days oftreatment. DNA fragmentation was determined after 48 hours of treatment.All data was collected from the same experiment.

FIG. 13 shows the apoptosis inducing properties of Compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof Compound E for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was 0.05 μM.

FIG. 14 shows the apoptosis inducing properties of Compound B. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof Compound B for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was approximately 175 μM.

TABLE 6 Apoptosis Inducing Activity Among a Series of Compounds Foldinduction at 100 μM Reference compounds Indomethacin <2.0 MY5445 4.7Sulindac sulfide 7.9 Exisulind <2.0 E4021 <2.0 Zaprinast <2.0 Sildenafil<2.0 EHNA <2.0 Test compounds A <2.0 B 3.4 C 5.6 D <2.0 E 4.6

In accordance with the fold induction protocol, supra, Compounds Athrough E were tested for apoptosis inducing activity, as reported inTable 6 above. Compounds B, C, and E showed significant apoptoticinducing activity, greater than 2.0 fold, at a dosage of 100 μM. Ofthese three compounds, at this dosage, only Compounds B and E did notinhibit COX but did inhibit cGMP-specific PDE.

The apoptosis inducing activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 7 below. HT-29cell were treated for 6 days with various inhibitors ofphosphodiesterase. Apoptosis and necrosis were determinedmorphologically after acridine orange and ethidium bromide labeling inaccordance with the assay described, supra. The data show cGMP-specificPDE inhibition represents a unique pathway to induce apoptosis inneoplastic cells.

TABLE 7 Apoptosis Induction Data for PDE Inhibitors Inhibitor ReportedSelectivity % Apoptosis % Necrosis Vehicle 8 6 8-methoxy-IBMX PDE1 2 1Milrinone PDE3 18 0 RO-20-1724 PDE4 11 2 MY5445 PDB5 80 5 IBMXNon-selective 4 13

V. Mammary Gland Organ Culture Model Tests

A. Introduction

Test compounds identified by the above methods can be tested forantineoplastic activity by their ability to inhibit the incidence ofpreneoplastic lesions in a mammary gland organ culture system. Thismouse mammary gland organ culture technique has been successfully usedby other investigators to study the effects of known antineoplasticagents such as NSAIDs, retinoids, tamoxifen, selenium, and certainnatural products, and is useful for validation of the methods used toselect cGMP-specific PDE inhibitors useful in the present invention.

For example, female BALB/c mice can be treated with a combination ofestradiol and progesterone daily, in order to prime the glands to beresponsive to hormones in vitro. The animals are sacrificed and thoracicmammary glands are excised aseptically and incubated for ten days ingrowth media supplemented with insulin, prolactin, hydrocortisone, andaldosterone. DMBA (7,12-dimethylbenz(a)anthracene) is added to medium toinduce the formation of premalignant lesions. Fully developed glands arethen deprived of prolactin, hydrocortisone, and aldosterone, resultingin the regression of the glands but not the premalignant lesions.

The test compound is dissolved in DMSO and added to the culture mediafor the duration of the culture period. At the end of the cultureperiod, the glands are fixed in 10% formalin, stained with alum carmine,and mounted on glass slides. The extent of the area occupied by themammary lesions can be quantitated by projecting an image of the glandonto a digitation pad. The area covered by the gland is traced on thepad and considered as 100% of the area. The space covered by each of theunregressed structures is also outlined on the digitization pad andquantitated by the computer.

The incidence of forming mammary lesions is the ratio of the glands withmammary lesions to glands without lesions. The incidence of mammarylesions in test compound treated glands is compared with that of theuntreated glands.

B. Activity in Mammary Gland Organ Culture Model

FIG. 15 shows the inhibition of premalignant lesions in mammary glandorgan culture by sulindac metabolites. Mammary gland organ cultureexperiments were performed as previously described (Mehta-and Moon,Cancer Research, 46: 5832-5835, 1986). The results demonstrate thatsulindac sulfoxide and exisulind effectively inhibit the formation ofpremalignant lesions, while sulindac sulfide was inactive. The datasupport the hypothesis that cyclooxygenase inhibition is not necessaryfor the anti-neoplastic properties of desired compounds.

CONCLUSIONS REGARDING PREFERRED PDE INHIBITORS

To identify cGMP-inhibiting compounds that are useful for treatingneoplasia in combination with paclitaxel, candidate cGMP-inhibitingcompounds can be selected by testing them as described above.

Qualitative data of various test compounds and the several protocols areshown in Table 8 below. The data show that exisulind, sulindac sulfide,MY5445, Compound B, and Compound E exhibit the appropriate activity tobe used with paclitaxel. In addition, those same compounds (except forsulindac sulfide and MY5445) are desirable because they lack COXinhibition activity. The activity of these compounds in the mammarygland organ culture validates the effectiveness of these compounds.

TABLE 8 Activity Profile of Various Compounds Mammary COX PDE GrowthApop- Gland Organ Compound Inhibition Inhibition Inhibition tosisCulture Exisulind − ++ ++ ++ +++ Sulindac ++++ +++ +++ +++ − sulfideMY5445 ++++ +++ +++ +++ + A − − +++ ++ ++ B − +++ +++ +++ ++ D − − ++ −− E − ++++ ++++ ++++ ++++ F − − ++ + − G − − +++ ++ +++ H − − ++ − −

Table 8. Code: Activity of compounds based on evaluating a series ofexperiments involving tests for maximal activity and potency.

−Not active

+Slightly active

++Moderately active

+++Strongly active

++++Highly active

COMBINATION TREATMENT WITH A PACLITAXEL DERIVATIVE AND A PDE INHIBITOR

The method of this invention involves treating a patient with neoplasiawith both a paclitaxel derivative and a cGMP-specific PDE inhibitor.There are a number of derivatives of paclitaxel or taxol. In thisregard, the two terms are used interchangeably herein. Variousderivatives of taxol (e.g., paclitaxel and docetaxel) are disclosedherein. Other taxol derivatives are disclosed in U.S. Pat. Nos.5,157,049, 4,814,470, 5,599,820, 5,705,508, 5,407,683, 5,475,011, and5,556,878, all of which are incorporated herein by reference. Suchcompositions collectively disclose non-limiting examples of “paclitaxelderivatives” as that term is used herein.

By treating a patient with this combination of pharmaceuticals,therapeutic results can be achieved that are not seen with either drugalone. As explained above, exisulind is one example of an appropriatecGMP-specific PDE inhibitor to be used in combination with a paclitaxelderivative in the practice of this invention. Exisulind inhibits bothPDE5 and the new cGMP-PDE, and treatment of neoplastic cells withexisulind results in growth inhibition and apoptosis. (See Table 8).

Exisulind and paclitaxel were tested together to determine theircombined effect on the growth of a tumor cell line. The ability of thecombination to inhibit tumor cell growth was tested by growing HT-29cells in exisulind (FGN-1) doses from 25 μM-400 μM in the presence ofpaclitaxel (taxol) doses varying from 0.1 nM to 10 nM. (See FIGS.15-20.) A standard SRB assay (see section III.A.) was performed todetermine the drugs' effect on cell growth.

The data show surprising and significant results on inhibition of cellgrowth after treatment with both exisulind and paclitaxel. FIG. 18, forexample, illustrates the effects on HT-29 cells grown in 100 μMexisulind and various doses of taxol. The percentage of growthinhibition with FGN-1 (exisulind) alone is shown in the first hatchedbar to the left, labeled FGN-1. The effect on cells grown in 100 μMFGN-1 combined with 0.1 μM taxol is shown in the next bar to the right,labeled 0.1. As illustrated in FIG. 18, the growth inhibition effects ofcombining taxol with a cGMP-specific PDE inhibitor, such as exisulind,are greater than the effects of either treatment alone. This combinedbenefit is most pronounced at exisulind doses of 25 μM to 100 μM (FIGS.16-18) and at taxol concentrations of less that 1 nM.

The method of this invention involves using combination therapy to treatpatients with neoplasia. Such combination therapy enhances the benefitto the patient without increasing harmful side effects. For example,exisulind is one cGMP-specific PDE inhibitor that can be used incombination with a paclitaxel derivative in this invention.

Exisulind has no significant side effects when administered at itsrecommended dose of 300-400 mg/day. When administered at doses higherthan the recommended therapeutic levels, treatment with exisulind canlead to elevated levels of liver enzymes. This effect is reversible, andliver enzymes return to normal levels when the administered dose ofexisulind returns to the traditionally recommended level or whentreatment is discontinued. The most serious side effect of taxol, on theother hand, is bone marrow suppression. Since the side effects of thetwo drugs do not overlap, a PDE inhibitor, such as exisulind, can beused in combination with taxol without increasing the harmful sideeffects of taxol.

A cGMP-specific PDE inhibitor and a paclitaxel derivative can be used incombination in at least two different ways. In the first method, thetraditionally recommended dose range of paclitaxel is reduced while itsbeneficial therapeutic effects are maintained and its side effects areattenuated. The second method uses the traditionally recommended doserange of paclitaxel with enhanced activity but without increasing itsside effects. In each of these methods, the patient is receiving bothdrugs, a PDE inhibitor and a paclitaxel derivative, eithersimultaneously or in succession, one after the other.

Taxol is most often used in the treatment of breast cancer and ovariancancer, but it has shown activity in cancers of the lung, head and neck,esophagus, and bladder as well. It is recommended that patientsreceiving therapy with a paclitaxel derivative be pretreated in order tolessen hypersensitivity reactions.

Pretreatment usually consists of corticosteroids such as dexamethazoneand antihistamines such as diphenhydramine.

For treatment of ovarian cancer, the common dose of taxol is 135 mg/m²or 175 mg/m² administered intravenously over 3 hours every 3 weeks. Forpatients with breast cancer, the recommended dose of taxol is 175 mg/m²administered intravenously over 3 hours every 3 weeks.

Docetaxel (Taxotere) is another antineoplastic agent of the taxoidfamily. Taxotere is most often used in the treatment of advanced breastcancer with a recommended dose of 60 to 100 mg/m² administeredintravenously over 1 hour every 3 weeks.

In one embodiment of this invention, the lower dose methodology,paclitaxel is administered at a dosage lower than 135 mg/m² incombination with a cGMP-specific PDE inhibitor. In another embodiment,docetaxel is administered at a dosage lower than 60 mg/m² in combinationwith a cGMP-specific PDE inhibitor. In this method, the combination oftherapies allows the benefits of paclitaxel derivative treatment to bemaintained while the side effects are reduced.

In the second embodiment, the dosage of paclitaxel is maintained at itstraditionally recommended range of 135 mg/m² to 175 mg/m² and isadministered in combination with a cGMP-specific PDE inhibitor. Inanother embodiment, docetaxel is administered at a dosage between 60mg/m² to 100 mg/m² in combination with a cGMP-specific PDE inhibitor.The combination, in this case, serves to increase the efficacy ofpaclitaxel derivative treatment without increasing its harmful sideeffects.

In the practice of this invention, a cGMP-specific PDE inhibitor and apaclitaxel derivative are used in combination such that the blood levelsof the inhibitor are at approximately the IC₅₀ value of the inhibitorfor growth inhibition. In the case of exisulind, it is recommended thatthe dose be about 200 to 400 mg/day administered between two to fourtimes a day.

In each of the aforementioned methodologies, the paclitaxel derivativeand the PDE inhibitor may be administered simultaneously or insuccession.

Paclitaxel derivatives are also used in combination with otherantineoplastic chemotherapeutics such as cisplatin (a platinumcoordination complex) or doxorubicin (an anthracyline antibiotic). Inthe practice of this invention, a cGMP-specific PDE inhibitor is used asan additional element of such combination therapies in order to increasethe efficacy of the treatment.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

We claim:
 1. A method of inhibiting the growth of neoplastic lesions ina patient comprising administering to the patient docletaxel and acGMP-specific phosphodiesterase inhibitor.